Plant inoculation method

ABSTRACT

A method for inoculating a plant with a nitrogen-fixing bacteria such as Gluconacetobacter diazotrophicus, said method comprising administering the nitrogen-fixing bacteria to a wound of a growing plant, for example to recently cut grass. Inoculation in this manner leads to enhanced growth characteristics including increased greenness of grass. Novel compositions suitable for use in the method are also described and claimed, together with kits for producing these.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/326,996, filed Jan. 17, 2017, which is a 371 of InternationalPatent Application No. PCT/GB2015/052170, filed Jul. 28, 2015, whichclaims the benefit of GB Application No. 1413333.4, filed Jul. 28, 2014,the disclosure of each is hereby incorporated by reference in itsentirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Jan. 11, 2017, is named 560170ST25.txt, and is 2000 bytes in size.

FIELD OF THE INVENTION

The present invention relates to a method for inoculating plants with anitrogen-fixing bacteria and to compositions and kits suitable for usein that method.

BACKGROUND TO THE INVENTION

The nitrogen-fixing bacterium Gluconacetobacter diazotrophicus,previously known as Acetobacter diazotrophicus (Gillis, M. et al. Int.J. Syst. Bacteriol. 39:361-364; 1989), was originally isolated fromwithin sugarcane roots and stems (Cavalcante, V. A., et al. (1988) PlantSoil Vol. 108, p. 23-31). It has been demonstrated by ¹⁵N2 incorporationthat G. diazotrophicus fixes nitrogen inside sugarcane plants (Sevilla,M. et al. Mol. Plant Microbe Interact. 14:358-366; 2001; Boddey, R. M.et al. Plant Soil 252:139-149; 2003) and that it has a capability toexcrete almost half of the fixed nitrogen in a form that is potentiallyavailable to plants (Cojho, E. H et al. Fed. Eur. Microbiol. Soc.Microbiol. Lett. 106:341-346; 1993). The bacterium invades between cellsof sugarcane root meristems and at emergence points of lateral rootscolonizing intercellularly, and also in the xylem, without nodulation(James, E. K. et al. J. Exp. Bot. 52:747-760; 2001). The conditionsunder which intracellular colonisation of Gd could occur enablingnon-nodular endosymbiotic nitrogen fixation has been demonstrated(EP-B-1422997 and Cocking, E. C., et al. (2006) In Vitro Cellular andDevelopmental Biology—Plant Vol. 42, No. 1, p 74-82). In particular, thebacteria are administered to the growth medium of the plant as the plantgrows on germination or within 7 days thereof.

WO2011/144741 suggests that bacteria such as Gd, may be injected intostems of sugarcane to enhance nitrogen-fixation. Clearly such atechnique is not one which could be applied in any large scaleagricultural operation.

The applicants have found that growing plants can be successfullyinoculated with nitrogen fixing bacteria.

SUMMARY OF THE INVENTION

According to the present invention there is provided a method forinoculating a plant with a nitrogen-fixing bacteria, said methodcomprising administering the nitrogen-fixing bacteria to a wound of agrowing plant.

DETAILED DESCRIPTION OF THE INVENTION

It has been found that when applied to a wound in particular to thesurface of a wound in plant tissue, subsequent plant growth is enhanced.For example the biomass or yield may be enhanced and/or, the number offlowers may be increased. This may be due to colonisation of the planttissue by the nitrogen-fixing bacteria in a similar manner to thatdescribed for instance in EP-B-1422997, although the fact that this mayoccur when applied in this manner is surprising. The nitrogen-fixingbacteria colonised within the plant tissue may provide a source ofintracellular nitrogen that enhances plant growth. Thus the method ofthe invention provides a useful means of administering a plant growthenhancing treatment to growing plants.

The nitrogen-fixing bacteria should suitably be one which may becomeintracellularly located within a plant cell. In a particular embodiment,this is the intracellulary colonising symbiotic nitrogen-fixing bacteriaGluconacetobacter diazotrophicus (Gd), for instance Gluconacetobacterdiazotrophicus strain IMI 504998 (formerly IMI 501986) or IMI 504958(formerly IMI 504853), both being deposited at CABI (UK). IMI 501986 isan accession number for a deposit with CABI that was made on 21 Sep.2012. There was then a conversion on 28 Jul. 2015 to a deposit under theBudapest Treaty, which was given the new accession number of 504998.Such strains are novel and form a further aspect of the invention.Alternatively, the nitrogen-fixing bacteria may be a species ofHerbaspirillum. Other nitrogen fixing bacteria include Azotobacter,Beijerinckia, Clostridium, Rhizobium, Klebsiella and Spirillum hpoferum.

In a particular embodiment, the nitrogen-fixing bacteria is administeredtogether or in combination with a strain of Terribacillus, as describedin the applicants co-pending International patent application whichclaims priority from British Patent Application No. 1400840.3. Theapplicants have found that such a strain may enhance the activity of thenitrogen-fixing bacteria. Suitable strains of Terribacillus includeTerribacillus saccharophilus, Terribacillus halophilus, Terribacillusgoriensis or Terribacillus aidingensis but in particular is a strain ofTerribacillus saccharophilus. The Terribacillus Terribacillus eitherseparately or in admixture with the nitrogen-fixing bacteria. TheTerribacillus may be in intimate admixture with the nitrogen-fixingbacteria, (and indeed, IMI501986 (now IMI 504998) has been classified asa consortium of Gd and Terribacillus) or it may be administered in aco-culture, or mixed culture form.

The wound may be a result of accidental or natural damage, whereupon theadditional nitrogen availability may facilitate repair growth. However,in a particular embodiment, the wound is the result of damage caused byactions such as mowing (amenity grass), cutting (silage and hay crops),ratooning (banana, pineapple, sugarcane, sorghum, rice, pigeonpea,cotton, Abaca, Ramie), pruning (fruit trees, vines), consumption bylivestock or by harvesting. Other processes, such as harrowing, in whichplants may be inadvertently or incompletely damaged, may not be suitablein some instances. In particular, the wound will be found in an‘above-ground’ part of the plant, such as leaves or stems.

Therefore, the method of the invention may further comprise apreliminary step of inflicting ‘damage’ on the plant, in particular bymowing, cutting, rationing, pruning or by harvesting. Thenitrogen-fixing bacteria is suitably applied within a relatively shorttime period of carrying out such actions, for instance, within 48 hours,for instance within 24 hours, such as within 10 hours and suitablywithin 1-2 hours of damage being inflicted on the plant.

Delivery of the bacteria is achieved by application of a suitableformulation to the wound area, in particular to the surface of thewound, in the form of a composition. The composition may be in the formof a liquid, gel, paste which may be applied directly or in dilutedform, or it may be in the form of a solid composition such as a powderor granule composition that will be dissolved in liquid such as waterbefore use. In solid compositions, the bacteria will generally be usedin dried form, for example in freeze-dried form, which arereconstitutable on addition of water. If desired, the bacteria may bemicroencapsulated using methods known in the art, in order to maintainhigh viability and stability of the bacteria.

In a particular embodiment, the composition is in a form suitable forspraying on the plants and thus will comprise a concentrate for dilutionwhich may be in the form of a liquid or solid, in particular in the formof a liquid, or it may comprise a dilute aqueous composition that may besprayed directly. Alternatively, the composition may be one in which thewound surface of a plant may be immersed by dipping for instance.

The amount of nitrogen-fixing bacteria that is administered in anyparticular case will vary depending upon factors such as the type ofseed being treated, the particular strain of nitrogen-fixing bacteriaused, the level of germination enhancement required and the method ofadministration, as well as the effect required. Typically however, asolution containing from 1 to 1×10⁷ bacteria per millilitre ofcomposition applied, for example from 10-10³ bacteria per millilitre ofcomposition for instance from 50-200 bacteria per millilitre ofcomposition such as 100 bacteria per millilitre of composition isadministered to the wounds of a plant. Such a solution may be obtainedby culturing the bacteria to a readily detectable level for example byexamining the optical density and then diluting the solutionaccordingly.

The applicants have found for instance that, in the case of certainbacteria, the effects on a property such as biomass, is affected by theamount of bacteria applied in a dose dependent manner. This means thatdifferent doses may be administered depending upon the aim of thetreatment. In the case of grasses for instance, it may be required thatbiomass is maximised in pasture grass, whereas in amenity or turf grass,slow growth may be preferable. In such cases, the amount of bacteriaadministered will be selected to provide optimum biomass production forthe target grass species, as exemplified below.

In a particular embodiment, the composition further comprises a nutrientfor the nitrogen-fixing bacteria, for example the composition maycomprise 3% w/v sucrose as described in EP-B-1422997.

The nitrogen-fixing bacteria may be the sole active component of thecomposition or it may be combined with additional agrochemically activecomponents such as insecticides, fungicides or plant growth regulatorsas required.

The composition may further comprise additives or excipients such asthickening agents, dispersants, diluents, humectants, solid carriersetc. as are known in the art.

In a particular embodiment, the composition further comprises apolysaccharide or an agriculturally acceptable surfactant or acombination of these.

In a particular embodiment, the composition further comprises anagriculturally acceptable surfactant. The presence of a surfactantensures that the composition is able to flow relatively freely over theentire surface of the wounds to facilitate entry of the nitrogen-fixingbacteria.

Suitable surfactants or detergents include non-ionic detergents such asthose sold under the trade name ‘Tween’®, for example Tween 80.

Tween 80 is a non-ionic detergent; 70% composed of the fatty acid oleicacid and the remainder a combination of linoleic, palmitic and stearicacids. The pH of a 1% solution is in the range of from 5.5-7.2. It iswidely used for emulsifying and dispersing substances in medicinal andfood products. It has little or no activity as an anti-bacterial agent(Dawson et al. (1986) Data for Biochemical Research, 3rd ed., OxfordUniversity Press (New York, N.Y.: 1986), p. 289).

The amount of surfactant administered to the plant wound should besufficient to produce an enhanced plant growth effect when incombination with the nitrogen-fixing bacteria (and optionally also apolysaccharide as described further below). This will vary dependingupon the various factors such as the particular surfactant, the type ofplant being treated, the nature of the wound, the particular strain ofnitrogen-fixing bacteria employed and the method of administration.However, typically, a composition comprising from 0.0005 to 10% v/v,such as from 0.0005 to 0.5% v/v, for instance from 0.0005 to 1% v/v,including from 0.0005 to 0.2% v/v for example from 0.0005 to 0.15% v/vsuch as about 0.1% v/v.

In a further embodiment, the composition comprises a polysaccharide.Suitable polysaccharides for use in the composition include hydrocolloidpolysaccharides derived from plant, animal or microbial sources.

In particular, these include exudate gum polysaccharides such as gumArabic, gum ghatti, gum karaya and gum tragacanth, cellulosicderivatives such as carboxymethylcellulose, methylcellulose,hydroxypropyl cellulose, hydroxypropyl methylcellulose ormicrocrystalline cellulose, starches and derivatives including, forinstance corn starch, tapioca starch, potato starch, rice starch, wheatstarch, and modified versions thereof such as pregelatinized starch,oxidized starch, ethylated starch, starch dextrins or maltodextrin,pectin, polysaccharides derived from seaweed such as agar, alginates,carrageenan, and fucellaran, seed gums such as guar gum and locust beangum, polysaccharides derived from microbial fermentation such as xanthangum and gellan gum, and nitrogen containing polysaccharides such aschitosan; or mixture of these.

In a particular embodiment, the polysaccharide is exudate gumpolysaccharide such as gum Arabic, gum ghatti, gum karaya or gumtragacanth. A particular example of the polysaccharide is gum Arabic.

Gum Arabica is a natural gum collected as exudates from differentspecies of Acacia trees (Fang et al. 2010 (2010) Biomolecules: 11,1398-1405); a complex polysaccharide it has been used extensively in awide range of industrial sectors including paint, glue, pharmaceuticals,textiles and food. Gum Arabic from the acacia tree is believed to be abranched polymer of galactose, rhamnose, arabinose, and glucuronic acidas the calcium, magnesium, and potassium salts with a mol. wt. ofapprox. 250,000. It has been shown (Badar, K. V. et al. (2011) RecentResearch in Science and Technology 3 (5) 6-7) to have an effect on seedgermination when seeds of certain plants are soaked in 1% solutions ofgum arabica for 24 hours prior to germination. Furthermore, WO02/058466reports that certain compositions comprising combinations ofpolysaccharides and peptides may increase crop yields.

The amount of polysaccharide administered to the plant wound should besufficient to produce an enhanced nitrogen-fixing effect when incombination with the nitrogen-fixing bacteria and optionally also asurfactant. This will vary depending upon the various factors such asthe particular polysaccharide used, the type of plant being treated, thenature of the wound, the particular strain of nitrogen-fixing bacteriaemployed and the method of administration. However, typically, acomposition comprising from 0.1 to 1% w/w, for example from 0.1 to 0.5%w/w such as about 0.3% w/w polysaccharide is used.

In one embodiment, the composition comprises both a polysaccharide andan agriculturally acceptable surfactant. It has been found that, in somecircumstances, these components enhance the effect of thenitrogen-fixing bacteria, and seem to work synergistically together toproduce a more significant enhancement. Plants treated with acomposition comprising these components may show increased growth asevidenced by increased dry weight of treated plants.

Novel compositions comprising the above-mentioned components form afurther aspect of the invention. Thus in a further aspect the inventionprovides an agriculturally acceptable composition comprising anitrogen-fixing bacteria, in particular Gluconacetobacterdiazotrophicus, and a polysaccharide, a surfactant or a combinationthereof.

The nitrogen-fixing bacteria are as described above, and in particularis Gluconacetobacter diazotrophicus are suitably present in the amountsdescribed above. Similarly, the polysaccharide is a polysaccharide asdescribed above, such as an exudate gum polysaccharide, for instance gumArabic, and this is included in the composition in an amount asdescribed above, for instance at a concentration of from 0.1 to 1% w/wpolysaccharide. In addition the surfactant is suitably a surfactant asdescribed above such as a non-ionic detergent, for instance surfactantthat is 70% composed of the fatty acid oleic acid and the remainder acombination of linoleic, palmitic and stearic acids. In a particularembodiment, the composition will comprise from 0.0005 to 10% v/vsurfactant for example from 0.0005 to 0.2% v/v surfactant.

In yet a further aspect, the invention provides a kit for preparing anagriculturally acceptable composition comprising a nitrogen-fixingbacteria. In such kits, the nitrogen-fixing bacteria, and in particularthe Gluconacetobacter diazotrophicus, may be held separately from othercomponents of the composition, for example in separate containers, or ina two-part pack or container. The nitrogen-fixing bacteria may befreeze-dried. The other components may be in the form of a concentrate,for ease of storage or transportation, ready for dilution with forexample, water, at the point of use. Concentrates of this nature willcontain the same components as the compositions listed above, but atgenerally higher levels. Thus, for example, a concentrate may containfrom 1 to 10% w/w, for example from 1 to 5% w/w such as about 3% w/wpolysaccharide, and a ten times dilution will result in the compositionsuitable for use in for example, the method of the invention. Similarly,the surfactant may be present in an amount of from 0.005 to 2% v/v inthe concentrate. Other components, such as for example, a nutrient forthe nitrogen-fixing bacteria is suitably including in the concentrate atthe required concentration.

Kits of this type may be used to produce a composition of the invention,which may be used directly. In particular any concentrate will bediluted with water to an appropriate volume, whereupon thenitrogen-fixing bacteria will be added thereto.

The invention enables intracellular nitrogen fixation bacteria to beapplied and delivered to a wide range of crops. In particular, these maybe perennial, biennial or persistent annuals including but not limitedto fruit trees and bushes (e.g. blueberries, raspberries and teaplants), vines, forage crops (alfalfa and grass for silage, hay ordirect consumption by livestock) amenity grass and hedges, forestry,horticulture and herbs (e.g. chives, asparagus, eggplant).

It has previously been reported that Gd may improve production ofsucrose-rich crops such as sugar beet or sugar cane (WO2010/022517).However, the applicants have found that using the treatment of theinvention, improvement is seen in non-sucrose-rich crops and these forma particular embodiment of the invention.

In a particular embodiment, the method and composition of the inventionis applied to grass such as amenity, turf or pasture grass, immediatelyor soon after mowing. This treatment leads to enhanced growth of thegrass as is evident by an increase in dry weight of inoculated versusun-inoculated grass. It appears that the nitrogen-fixing bacteria areable to enter the grass through the wounds resulting from the mowingprocedure, and colonise the grass plants intracellularly, leading toenhanced growth characteristics.

Furthermore, it has been found that colonization by Gd can increase thechlorophyll levels in plants and in particular in grass species such aspasture, amenity or turf grasses. As increase in chlorophyll is linkednot only to nitrogen content but also to the level of greenness of theplants, this property is highly desirable in applications such asamenity grass where high levels of greenness are beneficial.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be particularly described by way of example withreference to the accompanying diagrams in which:

FIG. 1 is a graph showing the mean dry weights (g) of un-inoculated andinoculated cut grass;

FIG. 2 is a graph showing the above ground dry weights of inoculated cutgrass treated with Gd and sucrose, Tween and/or Gum Arabic orcombinations thereof;

FIGS. 3A-3B illustrate an example of preparation of vegetative teapropagation,

FIG. 3A illustrates the removal of the cutting; FIG. 3B isdiagrammatical representation of sub-sections of each cutting taken forDNA isolation;

FIG. 4 shows an image of a gel of PCR products obtained from samples oftea plants which had been inoculated with Gd; all bands in controlplants were sequenced and confirmed as non-specific binding. Sequencedbands from inoculated plants were confirmed as Gluconacetobacterdiazotrophicus;

FIG. 5 is a graph showing the effects of various treatments on thebiomass of cut grass;

FIG. 6 shows the results of a test to determine the effect of Gd on thenumber of flower heads of grass; and

FIG. 7 is a graph showing the results of treatments with variouscompositions on the biomass of cut grass.

However, it will be apparent to one skilled in the art that the specificdetails are not required in order to practice the invention. Thefollowing descriptions of specific embodiments of the present inventionare presented for purposes of illustration and description. They are notintended to be exhaustive of or to limit the invention to the preciseforms disclosed. Obviously, many modifications and variations arepossible in view of the above teachings. The embodiments are shown anddescribed in order to best explain the principles of the invention andits practical applications, to thereby enable others skilled in the artto best utilize the invention and various embodiments with variousmodifications as are suited to the particular use contemplated.

Example 1

Application to Cut Grass

Methodology

Culture of G. diazotrophicus:

G. diazotrophicus strain IMI 501986 (now IMI 504998) with the pRGS561plasmid expressing GUS, were cultured on ATGUS medium, [0.8% (w/v) agar,yeast extract (2.7 g 1⁻¹), glucose (2.7 g 1⁻¹), mannitol (1.8 g 1⁻¹),MES buffer (4.4 g 1⁻¹), K₂HPO₄ (4.8 g 1⁻¹), and KH₂PO₄ (0.65 g 1⁻¹), pH6.5] as required. Expression of the b-glucuronidase (gusA) gene wastested by plating on ATGUS medium containing X-Gluc(5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid cyclohexylammoniumsalt) at 50 mg 1⁻¹; the formation of dark blue colonies indicated gusAgene expression. Inoculation procedures:

An aqueous suspension of the G. diazotrophicus was prepared to give anoptical density at 600 nm of 1.1, c. 10⁹ colony forming units (CFU) permilliliter. The number of CFU was determined by serial dilution, platingon ATGUS medium (with antibiotics as appropriate) and counting bacterialcolonies after 4d incubation in Petri dishes (28° C., dark). Thesuspension was diluted to 10⁻⁴ to produce a solution containingapproximately 100 bacteria per ml ready for spraying as described below.

A standard weight of 0.5 g of grass Lolium perenne variety Cassiopeiaseeds were sown in seedling trays of John Innes No. 1 compost andlightly covered with compost.

The individual trays were placed in larger trays and provided withadequate water in a growth room at 21° C./15° C. day/night 16/8 h cyclefor 20 days. After which the grass was cut at a height of 2 cm abovesoil level using scissors (clippings were removed) and the followingtreatments were applied using a domestic handheld mist sprayer:

Experiment 1. Treatments

Control of water+3% sucrose

Gd+water+3% sucrose

Experiment 2 Treatments

Gd+water

Gd+water+3% sucrose

Gd+water+0.1% Tween

Gd+water+0.3% Gum Arabic

Gd+water+3% sucrose+0.1% Tween

Gd+water+3% sucrose+0.3% Gum Arabic

Gd+water+3% sucrose+0.1% Tween+0.3% Gum Arabic

Dry Weight of Germinated Seedlings

The seedlings were removed from the agar with forceps and all remainingagar washed from the roots. Each seedling was placed in a paper bag andplaced in an oven 80° C. for 48 hours and then weighed.

Results from Experiments 1 and Experiment 2 are shown in FIGS. 1 and 2respectively.

The results in FIG. 1 show a significant increase in the mean dry weightof the grass (0.09676 g for un-inoculated and 0.1276 g for inoculatedgraph). These dry weights were significantly different at P<0.01. Thus,inoculation in this manner clearly leads to a significant enhancement ofgrowth.

These results shown in FIG. 2 show a significant difference (P<0.001)between Gd/S/T/GA and the next highest dry weight (Gd/T) and Gd/S/T,demonstrating a synergistic effect of the combination of threecomponents. Gd and Gd/S are not significantly different at P=0.05.

Example 2

Colonisation of Tea (Camellia sinensis) BY Gluconacetobacterdiazotrophicus (Azoticus)

Vegetative Reproduction from a Stem Cutting

The standard means of vegetative propagation of tea clones is asingle-leaf cutting. From larger stems comprising of approximately fourto six nodes and a shoot tip, sections of stem and leaf were selectedbased upon health of the tissue (i.e. free of insects and diseases). Thesection chosen for the cutting was between red and green wood asrecommended by Yamasaki et al. Soil and Crop Management, (2008) SCM-23).Recently matured shoots containing slightly reddened bark adjacent tomature leaves with actively breaking axillary buds have been found toresult in the best rooting success.

From the preferred sections, a sample was selected comprising of a 3-5cm section of stem and one healthy leaf. Each stem section was excisedusing a diagonal cut (1) approximately 0.5 cm above the leaf (2) andanother diagonal cut below the leaf around an internode (3) avoidingpinching or bruising of the wound site (See FIG. 3A.)

The bottom of each tea stem cutting was dipped into 1% indole-butyricacid solution and placed into individual pots; the cutting planted withthe stem straight of slightly slanted so that the leaf does not touchthe soil. Each pot contained sand and John Innes number 1 cutting mix ina 4:1 ratio, saturated with water. To the cut top surface of eachcutting either 20 μl of water, or 20 μl of Gd at 2.5×10⁵ cfu/ml in waterwas applied, and the humidity of each sample maintained by covering eachpot with a plastic sheet and sprayed lightly with water.

Following 3 months growth, and in order to confirm successfulcolonisation of the stem cuttings with Gd, uninoculated and inoculatedstem cuttings were removed from the pots. Each cutting was sub-dividedinto sections which were (a) the top of shoot, including inoculationsite (4) in (FIG. 3B), (b) the nodal section (5) in FIG. 3B, and (C) thelower stem section including any root tissue (6) in FIG. 3B. Thesesections where then snap frozen in liquid nitrogen.

DNA isolation from each section of cutting (i.e. 4, 5 and 6 in FIG. 3B)was carried out using TRIzol reagent according to the manufacturer'sprotocol and PCR performed. The PCR reaction carried out was a two-stepreaction as described by Tian et. al., (2009); the first step usingGDI-25F (5′-TAGTGGCGGACGGGTGAGTAACG-3′; SEQ ID NO:1) and GDI-923R(5′-CCTTGCGGGAAACAGCCATCTC-3′; SEQ ID NO:2) which amplified an 899 bpproduct containing the amplicon of primers GDI139F(5′TGAGTAACGCGTAGGGATCTG-3′; SEQ ID NO:3) and GDI916R(5′-GGAAACAGCCATCTCTGACTG-3′; SEQ ID NO:4), the latter designed basedupon 16S rDNA sequence information available in the GenBank database.After an initial denaturation step at 95° C. for 3 minutes, thefollowing temperature profile was executed 32 times; denaturation for 20seconds at 95° C., annealing for 45 seconds at 55° C., and extension for20 seconds at 72° C., with a final extension step of 5 minutes at 72° C.One microliter of this PCR product was then taken and used as templatefor the second step of the PCR using GDI39F and GDI916R. Modificationsto the parameters in the second round included increasing the annealingtemperature to 62° C. for 15 seconds, and increasing the cycle number to39. The PCR amplification products were analysed on a 1% agarose gelstained with ethidium bromide as well as sequenced to confirm identityof product (FIG. 4 ).

Interestingly, AzGd was not detected in section 4 of the tea cuttingsuggesting that AzGd moved basipetally from the wound site followinginoculation, being detected in sections 5 and 6 respectively.

Sequencing and subsequent BLAST results provided confirmation that thebands seen in section 1 of control plants were as a result ofnon-specific binding of the primer sets used, with the 4 bands observedin sections 2 and 3 of inoculated tissue being identified asGluconacetobacter diazotrophicus Pal5 (at 100% identification, 86% querycover and an E-value of 7e-04). The results suggest that AzGd althoughat a low copy number in the inoculated tissue did successfully coloniseCamellia sinensis following inoculation of the wound site. This ispossibly the first example of colonisation of a perennial plant by Gd.

Example 3

Investigation of Effect of Treatment on Grass Biomass

Grass was grown in a plant growth chamber (Fitotron®) (23° C./15° C. at65% humidity) in seed trays using John Innes No. 1 compost, for 2 weeks.It was then cut to a height of 8 cm and immediately sprayed with 10 mlof treatment as set out below using a domestic sprayer.

Treatments

1. Water

2. 3% sucrose+0.1% Tween+0.3% Gum Arabic

3. Water+Gd (2.5×10⁵ cfu/ml)

4. Water+3% sucrose+0.1% Tween+0.3% Gum Arabic+Gd (2.5×10³ cfu/ml)

5. Water+3% sucrose+0.1% Tween+0.3% Gum Arabic+Gd (2.5×10⁴ cfu/ml)

6. Water+3% sucrose+0.1% Tween+0.3% Gum Arabic+Gd (2.5×10⁵ cfu/ml)

7. Water+3% sucrose+0.1% Tween+0.3% Gum Arabic+Gd (2.5×10⁶ cfu/ml)

8. Water+3% sucrose+0.1% Tween+0.3% Gum Arabic+Gd (2.5×10⁷ cfu/ml)

The grass was returned to the Fitotron for a further 2 weeks undersimilar growth conditions. 5 plants, chosen at random, were cut at thesoil level to form one sample and weighed. This was repeated a furtherfive times to give six samples in total for each treatment.

These samples were dried in the oven for 48 hours and weighed.

The results are shown in FIG. 5 . These results show that, provided somesucrose is present to support the growth of Gd, the biomass of the grassincreased with the addition of Gd depending upon the formulation.Furthermore, the increase was dose dependent, with an optimum growthbeing observed at 2.5×10⁶ cfu/ml. Such a dose may therefore bebeneficial if the grass treated is pasture grass where maximisingbiomass is beneficial. However, if the grass treated is amenity or turfgrass, lower biomass with enhanced greenness may be beneficial in thatit may improve appearance without increasing the need for furthercutting or mowing. In this case, a dosage of either less than or greaterthan 2.5×10⁶ cfu/ml may be used.

Example 4

Field Trial

A formulation comprising water+3% sucrose+0.1% Tween+0.3% gum Arabic+Gd(2.5×10⁵ cfu/ml) was applied to a single 1 m² cut grass plot(established Lolium perenne turf) relative to an 1 m² uninoculated cutgrass plot treated with water only (control).

The formulation and water were applied, within 30 minutes of the grassbeing freshly mown, using a household mist sprayer to run-off. Thecontrol plot was protected from the treatment plot by a plastic screen.The application was made late afternoon in still air.

The 1 m² plots were subsampled using a 20 cm squared wire quadrant bycounting the number of fully extended and fully formed flowering heads.

The results from each 20 cm square within each plot was averaged and theresults are shown in FIG. 6 . It is clear that the Gd treatment, appliedin this way, substantially impacted on flower growth.

Example 5

Comparison of Components of Composition

The method of Example 3 was repeated using various compositionsincluding individual components of the composition used in thatexperiment. Specifically, the compositions used in this experiment wereas follows:

Treatments

1. Water

2. Water+Gd (2.5×10⁵ cfu/ml)

3. Water+3% sucrose+0.1% Tween+0.3% Gum Arabic+Gd (2.5×10⁵ cfu/ml)

4. 0.3% Gum Arabic+Gd (2.5×10⁵ cfu/ml)

5. 3% Sucrose+Gd (2.5×10⁵ cfu/ml)

6. 0.1% Tween+Gd (2.5×10⁵ cfu/ml)

AberGlyn grass was grown for 2 weeks in John Innes No. 1 soil in a plantgrowth chamber (Fitotron®) at 23/15° C., 80% humidity. The grass was cutto a height of 8 cm with scissors, the cuttings removed and the grassimmediately sprayed with the 10 ml treatment using a domestic sprayer.The grass was returned to plant growth chamber for a further two weeks.

Five plants were chosen at random from the tray and pooled together tomake one sample and weighed. This was repeated a further five times so atotal of six samples were taken per treatment. Grass was dried for 48hours at 80° C. and then weighed.

The results are shown in FIG. 7 . This experiment shows that thecomponent used does have an effect on the growth of the grass. In thisexample, the surfactant gave the greatest increase in dry weight. GumArabic showed a marginal improvement only over the control, possibly dueto the fact that the surfactant may be required to assist in the spreadof the Gd on the plant and helps the liquid enter the wounds of thegrass (although on this occasion, the combination did not show theexpected an improvement). Again the water+Gd treatment was similar tothe control so indicates that Gd needs the addition of at least some ofthese components to colonise the wounds.

What is claimed is:
 1. A method comprising delivering to a plant or seeda composition comprising Gluconacetobacter diazotrophicus, apolysaccharide, and a nonionic detergent.
 2. A plant or seed comprisinga composition comprising Gluconacetobacter diazotrophicus, apolysaccharide, and a nonionic detergent.
 3. A method according to claim1, wherein Gluconacetobacter diazotrophicus is a strain deposited withCABI in the United Kingdom under deposit accession number IMI 504958 orunder deposit accession number IMI 504998, or is a nitrogen-fixingvariant of such a strain.
 4. A plant or seed according to claim 2,wherein Gluconacetobacter diazotrophicus is a strain deposited with CABIin the United Kingdom under deposit accession number IMI 504958 or underdeposit accession number IMI 504998, or is a nitrogen-fixing variant ofsuch a strain.
 5. A composition or kit comprising Gluconacetobacterdiazotrophicus, a polysaccharide and a non-ionic detergent, wherein theGluconacetobacter diazotrophicus is a strain deposited with CABI in theUnited Kingdom under deposit accession number IMI 504958 or underdeposit accession number IMI 504998, or is a nitrogen-fixing variant ofsuch a strain.
 6. A plant spray comprising Gluconacetobacterdiazotrophicus, a polysaccharide and a non-ionic detergent.